BACKGROUND:
Modeling the human blood-brain barrier (BBB) is limited by the lack of robust protocols to generate induced pluripotent stem cell (iPSC)–derived brain micro... More
BACKGROUND:
Modeling the human blood-brain barrier (BBB) is limited by the lack of robust protocols to generate induced pluripotent stem cell (iPSC)–derived brain microvascular endothelial cells (BMECs). Current methods generate cells that do not fully recapitulate key BMEC functions or the brain endothelial transcriptome identity.
METHODS:
To address this gap, we combined directed differentiation of human iPSCs into BBB-primed endothelial cells with overexpression of FOXF2 (forkhead box F2) and ZIC3 (zic family zinc finger 3), transcription factors critical for BMEC identity, to generate reprogrammed BMECs (rBMECs) from 3 iPSC lines. We performed immunofluorescence, functional analyses, and bulk RNA sequencing to characterize these cells. We cocultured rBMECs with iPSC-derived astrocytes and pericytes in the MIMETAS microfluidics platform to assess how 3-dimensional culture influences their BBB properties. Finally, we generated rBMECs expressing familial Alzheimer disease mutation APP V717I to elucidate how this genetic variant affects barrier properties compared with exposure to oAβ42 (oligomeric amyloid-β [1-42] peptide).
RESULTS:
Transcriptomic and functional analyses show that rBMECs express a subset of the BBB transcriptome and exhibit stronger paracellular barrier properties, lower caveolar-mediated transport, and comparable PGP (P-glycoprotein) activity compared with primary human BMECs. rBMECs interact with human iPSC–derived pericytes and astrocytes to form a 3D neurovascular system in the MIMETAS microfluidics platform with robust BBB properties. Finally, APP V717I rBMECs show decreased barrier integrity and upregulation of inflammatory markers. In contrast, treatment of control rBMECs with oAβ42 increases inflammatory markers, but does not alter barrier integrity.
CONCLUSIONS:
This protocol generates rBMECs with strong BBB properties and a brain-specific transcriptome signature. In addition, the iPSC-derived 3D neurovascular unit system shows some similar properties to the in vivo human BBB. Finally, familial Alzheimer disease mutation APP V717I alters several BBB-related properties of rBMECs and their inflammatory state, independent of Aβ42 (amyloid-β [1-42] peptide). Less
Ménière’s disease (MD) is thought to involve dysfunction of the blood–labyrinth barrier, but circulating mechanisms of endothelial injury remain poorly understood. ... More
Ménière’s disease (MD) is thought to involve dysfunction of the blood–labyrinth barrier, but circulating mechanisms of endothelial injury remain poorly understood. The present study investigated whether cell-free DNA (cfDNA) and inflammatory mediators in plasma contribute to vascular stress and barrier disruption in MD. cfDNA levels were significantly elevated in plasma from patients compared with plasma from healthy controls. Exposure of primary human stria vascularis endothelial cell monolayers to plasma from MD patients led to decreased transepithelial electrical resistance and a significant increase in FITC-dextran permeability, indicating impaired barrier function. MD plasma also induced higher lactate dehydrogenase release and pronounced F-actin disorganization with reduced syndecan-1 expression, consistent with endothelial cytotoxicity and glycocalyx degradation. DNase I partially reversed these effects, implicating extracellular DNA as a key driver. Furthermore, IL-1β, CCL3 (MIP-1α), and CCL27 were elevated in MD plasma. Collectively, our data support a model in which cfDNA and inflammatory mediators cooperatively induce endothelial injury, cytoskeletal remodeling, and glycocalyx shedding, leading to blood–labyrinth barrier weakening. Targeting extracellular DNA or glycocalyx preservation may represent a novel strategy to protect inner ear vascular integrity and modify disease progression in MD, and cfDNA-related readouts may be promising biomarkers of endothelial damage. Less
The blood vessels of the central nervous (CNS) system form a tight, protective blood-brain barrier (BBB). This barrier is essential for healthy CNS function but also pose... More
The blood vessels of the central nervous (CNS) system form a tight, protective blood-brain barrier (BBB). This barrier is essential for healthy CNS function but also poses a hurdle in the treatment of increasingly common neurological disorders. Additionally, BBB dysfunction is a hallmark of many neurological diseases, further emphasizing a need for a better understanding of BBB function in health and disease. We present a human self-assembling 3D model of the BBB in a microfluidic cell culture platform that allows culture of 48 models in parallel on one tissue culture plate. Human brain microvascular endothelial cells, pericytes, and astrocytes form highly reproducible BBB vascular networks under unidirectional perfusion and remain viable for a minimum of 14 days. Immunostaining reveals close cell-cell interactions with pericytes and astrocyte endfeet in direct contact with the brain microvasculature. Compared to endothelial monocultures, co-cultures with astrocytes or pericytes result in improved barrier function, lower vessel diameters, increased branching, and alignment of the vessels in the direction of fluid flow. These results were most pronounced in tri-cultures containing all three cell types. Unlike similar models previously reported, this brain microvasculature model allows for unidirectional perfusion without the need for pumps and syringes. Combined with its high-throughput nature, this feature renders the model suitable for studies of BBB function in health and disease, and assessment of potential BBB restorative therapies. Less
Longitudinal live cell imaging is valuable for characterizing dynamic morphological and phenotypic changes in biological systems. How-ever, conventional approaches rely o... More
Longitudinal live cell imaging is valuable for characterizing dynamic morphological and phenotypic changes in biological systems. How-ever, conventional approaches rely on manual microscope operation, which is labor-intensive, limits imaging frequency, and disrupts the cellular environment. These constraints reduce scalability, increase experimental variability, and restrict both the duration and temporal resolution of continuous imaging. Although auto-mated imaging platforms partially address these limitations, existing solutions are often constrained by the cost, footprint, and inflexibility of in-incubator microscopes or stage-top incubators. Here, we present an automated in-incubator epifluorescence micro-scope designed for long-term operation. The system features a modular architecture with optional multi-fluorescence imaging, automated plate scanning, configurable light sources, and compatibility with multiple plate formats, including integration with fluidic automation devices. By positioning the light sources and control electronics outside the incubator, the platform improves thermal stability and long-term operational reliability. This approach enables continuous, high-frequency imaging over extended durations, providing a source of rich data for quantifying time-dependent tissue phenotypes, morphological remodeling, and transient biological processes. Less
Hematogenous metastasis is the leading cause of cancer mortality, with dysfunction of pericytes, key components of tumor vessels, playing a central role in facilitating m... More
Hematogenous metastasis is the leading cause of cancer mortality, with dysfunction of pericytes, key components of tumor vessels, playing a central role in facilitating metastatic spread. Although anti-pericyte therapies are gaining recognition for treating metastasis, current strategies that directly eliminate tumor pericytes (TPCs) may increase vascular leakiness, which paradoxically promotes further metastasis. Here, we identify a TPC-specific transcription factor heterodimer, TCF21-TCF3, which drives metastasis by enhancing collagen hydroxylation and extracellular matrix deposition. Based on the TCF21 residues that interact with TCF3, we rationally design a peptide to disrupt their dimerization and downregulate TCF21-TCF3-dependent collagen deposition. Notably, in murine models of colorectal cancer and osteosarcoma, the TCF21-derived peptide significantly inhibits metastasis by restoring the physiological gatekeeper function of pericytes on vessels, offering a potential therapeutic strategy to target TPCs and suppress metastasis. Our findings reveal a TPC-specific transcription factor heterodimer and provide a promising pericyte-targeting strategy for preventing hematogenous metastasis. Less
Pericytes line the microvasculature throughout the body and play a key role in regulating blood flow by constricting and dilating vessels. However, the biophysical mechan... More
Pericytes line the microvasculature throughout the body and play a key role in regulating blood flow by constricting and dilating vessels. However, the biophysical mechanisms through which pericytes transduce microenvironmental chemical and mechanical cues to mediate vessel diameter, thereby impacting oxygen and nutrient delivery, remain largely unknown. This knowledge gap is clinically relevant as numerous diseases are associated with the aberrant contraction of pericytes, which are unusually susceptible to injury. Here, we report the development of a high-throughput hydrogel-based pericyte contraction cytometer that quantifies single-cell contraction forces from murine and human pericytes in different microvascular microenvironments and in the presence of competing vasoconstricting and vasodilating stimuli. We further show that murine pericyte survival in hypoxia is mediated by the mechanical microenvironment and that, paradoxically, pre-treating pericytes to reduce contraction increases hypoxic cell death. Moreover, using the contraction cytometer as a drug-screening tool, we found that cofilin-1 could be applied extracellularly to release murine pericytes from hypoxia-induced contractile rigor mortis and, therefore, may represent a novel approach for mitigating the long-lasting decrease in blood flow that occurs after hypoxic injury. Less
Background: Brain pericytes ensheathe the endothelium and contribute to formation and maintenance of the blood-brain-barrier. Additionally, pericytes are involved in seve... More
Background: Brain pericytes ensheathe the endothelium and contribute to formation and maintenance of the blood-brain-barrier. Additionally, pericytes are involved in several aspects of the CNS immune response including scarring, adhesion molecule expression, chemokine secretion, and phagocytosis. In vitro cultures are routinely used to investigate these functions of brain pericytes, however, these are highly plastic cells and can display differing phenotypes and functional responses depending on their culture conditions. Here we sought to investigate how two commonly used culture media, high serum containing DMEM/F12 and low serum containing Pericyte Medium (ScienCell), altered the phenotype of human brain pericytes and neuroinflammatory responses.
Methods: Pericytes were isolated from adult human brain biopsy tissue and cultured in DMEM/F12 (D-pericytes) or Pericyte Medium (P-pericytes). Immunocytochemistry, qRT-PCR, and EdU incorporation were used to determine how this altered their basal phenotype, including the expression of pericyte markers, proliferation, and cell morphology. To determine whether culture media altered the inflammatory response in human brain pericytes, immunocytochemistry, qRT-PCR, cytometric bead arrays, and flow cytometry were used to investigate transcription factor induction, chemokine secretion, adhesion molecule expression, migration, phagocytosis, and response to inflammatory-related growth factors.
Results: P-pericytes displayed elevated proliferation and a distinct bipolar morphology compared to D-pericytes. Additionally, P-pericytes displayed lower expression of pericyte-associated markers NG2, PDGFRβ, and fibronectin, with notably lower αSMA, CD146, P4H and desmin, and higher Col-IV expression. Nuclear NF-kB translocation in response to IL-1β stimulation was observed in both cultures, however, P-pericytes displayed elevated expression of the transcription factor C/EBPδ, and lower expression of the adhesion molecule ICAM-1. P-pericytes displayed elevated phagocytic and migratory ability. Both cultures responded similarly to stimulation by the growth factors TGFβ1 and PDGF-BB.
Conclusions: Despite differences in their phenotype and magnitude of response, both P-pericytes and D-pericytes responded similarly to all examined functions, indicating that the neuroinflammatory phenotype of these cells is robust to culture conditions.
Keywords: Blood–brain barrier; Growth factor; Inflammation; Migration; Phagocytosis; Proliferation. Less
Muscle-resident PDGFRβ+ cells, which include pericytes and PW1+ interstitial cells (PICs), play a dual role in muscular dystrophy. They can either undergo myogenesis to ... More
Muscle-resident PDGFRβ+ cells, which include pericytes and PW1+ interstitial cells (PICs), play a dual role in muscular dystrophy. They can either undergo myogenesis to promote muscle regeneration or differentiate into adipocytes and other cells to compromise regeneration. How the differentiation and fate determination of PDGFRβ+ cells are regulated, however, remains unclear. Here, by utilizing a conditional knockout mouse line, we report that PDGFRβ+ cell-derived laminin inhibits their proliferation and adipogenesis, but is indispensable for their myogenesis. In addition, we show that laminin alone is able to partially reverse the muscle dystrophic phenotype in these mice at the molecular, structural and functional levels. Further RNAseq analysis reveals that laminin regulates PDGFRβ+ cell differentiation/fate determination via gpihbp1. These data support a critical role of laminin in the regulation of PDGFRβ+ cell stemness, identify an innovative target for future drug development and may provide an effective treatment for muscular dystrophy. Less
Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB) which leads to infiltration of blood cells, an inflammatory response, and neuronal... More
Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB) which leads to infiltration of blood cells, an inflammatory response, and neuronal cell death, resulting spinal cord secondary damage. Retinoic acid (RA) has a neuroprotective effect in both ischemic brain injury and SCI, however the relationship between BSCB disruption and RA in SCI is still unclear. In this study, we demonstrated that autophagy and ER stress are involved in the protective effect of RA on the BSCB. RA attenuated BSCB permeability and decreased the loss of tight junction (TJ) molecules such as P120, β-catenin, Occludin and Claudin5 after injury in vivo as well as in Brain Microvascular Endothelial Cells (BMECs). Moreover, RA administration improved functional recovery in the rat model of SCI. RA inhibited the expression of CHOP and caspase-12 by induction of autophagic flux. However, RA had no significant effect on protein expression of GRP78 and PDI. Furthermore, combining RA with the autophagy inhibitor chloroquine (CQ) partially abolished its protective effect on the BSCB via exacerbated ER stress and subsequent loss of tight junctions. Taken together, the neuroprotective role of RA in recovery from SCI is related to prevention of of BSCB disruption via the activation of autophagic flux and the inhibition of ER stress-induced cell apoptosis. These findings lay the groundwork for future translational studies of RA for CNS diseases, especially those related to BSCB disruption. Less
We have recently described the response of human brain pericytes to lipopolysaccharide (LPS) through toll-like receptor 4 (TLR4). However, Gram-negative pathogen-associat... More
We have recently described the response of human brain pericytes to lipopolysaccharide (LPS) through toll-like receptor 4 (TLR4). However, Gram-negative pathogen-associated molecular patterns include not only LPS but also peptidoglycan (PGN). Given that the presence of co-purified PGN in the LPS preparation previously used could not be ruled out, we decided to analyse the expression of the intracellular PGN receptors NOD1 and NOD2 in HBP and compare the responses to their cognate agonists and ultrapure LPS. Our findings show for the first time that NOD1 is expressed in pericytes, whereas NOD2 expression is barely detectable. The NOD1 agonist C12-iE-DAP induced IL6 and IL8 gene expression by pericytes as well as release of cytokines into culture supernatant. Moreover, we demonstrated the synergistic effects of NOD1 and TLR4 agonists on the induction of IL8. Using NOD1 silencing in HBP, we showed a requirement for C12-iE-DAP-dependent signalling. Finally, we could discriminate NOD1 and TLR4 pathways in pericytes by pharmacological targeting of RIPK2, a kinase involved in NOD1 but not in TLR4 signalling cascade. p38 MAPK and NF-κB appear to be downstream mediators in the NOD1 pathway. In summary, these results indicate that pericytes can sense Gram-negative bacterial products by both NOD1 and TLR4 receptors, acting through distinct pathways. This provides new insight about how brain pericytes participate in the inflammatory response and may have implications for disease management. Keywords: NOD1; TLR4; inflammation; lipopolysaccharide; peptidoglycan; pericyte; vascular biology. Less
Background Human cytomegalovirus (HCMV) is the leading infectious cause of vision loss among congenitally infected children. Retinal pericytes play an essential role in m... More
Background Human cytomegalovirus (HCMV) is the leading infectious cause of vision loss among congenitally infected children. Retinal pericytes play an essential role in maintaining retinal vascular and endothelial cell proliferation. However, the role of retinal pericytes in ocular HCMV pathogenesis is unknown. Methods Retinal pericytes were exposed to clinical (SBCMV) and lab strains of HCMV; infectivity was analyzed by microscopy, immunofluorescence and qRT-PCR (reverse transcription polymerase chain reaction). Cytokine expression was examined by Luminex assay. Recombinant HCMV-GPF was used to examine viral replication kinetics. A Tricell culture model of the inner blood-retinal barrier (IBRB) was examined for cell type infectivity using immunohistochemistry. Results Retinal pericytes expressed the biomarker neuron-glial antigen 2. Antigenic expression profiles for several cytoskeletal, cell adhesion and inflammatory proteins were shared by both retinal and brain pericytes. Infected pericytes showed cytomegalic cytopathology and expressed mRNAs for the major immediate protein (MIE) and HCMV phosphorylated envelop protein 65. qRT-PCR analysis showed full lytic replication of HCMV in retinal pericytes. Pericytes exposed to SBCMV for 9 days expressed higher levels of vascular endothelial cell growth factor mRNA compared to controls. Luminex analysis of supernatants from SBCMV-infected retinal pericytes had increased levels of macrophage inflammatory protein-1α, beta-2 microglobulin (B2-m), matrix metalloproteinase-3 and -9 (MMP3/9), and lower levels of IL-6 and IL-8 compared to controls. At 24 hours post infection, pericytes expressed higher levels of IL-8, TIMP-1 (tissue inhibitor of metalloproteinase-1), and RANTES (regulated upon activation normal T cell-expressed and presumably secreted) but lower levels of MMP9. Time course analysis showed that both brain and retinal pericytes were more permissive for HCMV infection than other cellular components of the BBB (blood-brain barrier) and IBRB. Using a Tricell culture model of the IBRB (retinal endothelial, pericytes, Müller cells), retinal pericytes were most permissive for SBCMV infection. SBCMV infection of this IBRB Tricell mixture for 96 hours resulted in increased levels of IL-6, MMP9, and stem cell factor with a concomitant decrease in granulocyte-macrophage colony-stimulating factor and TNF-alpha. Conclusion In retinal pericytes, HCMV induces proinflammatory and angiogenic cytokines. In the IBRB, pericytes likely serve as an amplification reservoir which contributes to retinal inflammation and angiogenesis. Less
Rationale: Pericytes are perivascular cells localized to capillaries that promote vessel maturation and their absence can contribute to vessel loss. Whether impaired peri... More
Rationale: Pericytes are perivascular cells localized to capillaries that promote vessel maturation and their absence can contribute to vessel loss. Whether impaired pericyte interaction with endothelial cells contributes to small vessel loss in pulmonary arterial hypertension (PAH) is unclear. Objective: To measure the ability of PAH pericytes to associate with pulmonary microvascular endothelial cells (PMVECs) in vitro and in vivo. Methods and Results: Using 3G5 specific IgG-coated magnetic beads, we isolated pericytes from lungs of healthy and PAH patients followed by lineage validation. When seeded with healthy PMVECs, PAH pericytes failed to associate with endothelial tubes, resulting in smaller vascular networks compared to those seen with healthy pericytes. Following demonstration of abnormal polarization towards endothelium via live imaging and wound-healing studies, we screened PAH pericytes for abnormalities in the Wnt/planar cell polarity (PCP) pathway, which has been shown to regulate cell motility and polarity in the pulmonary vasculature. We found that PAH pericytes have reduced expression of frizzled 7 (Fzd7) and cdc42, two genes critical for Wnt/PCP activation. Simultaneous knockdown of Fzd7 and cdc42 in healthy pericytes resulted in reduced motility and polarization towards PMVECs both in vitro and in a murine model of angiogenesis, whereas restoration of both genes in PAH pericytes resulted in improved endothelial-pericyte association and larger vascular networks. Conclusions: These studies suggest that the motility and polarity of pericytes during pulmonary angiogenesis is regulated by Wnt/PCP activation. Therapies targeting Wnt/PCP in pericytes could help prevent vessel loss in PAH. Less
Retinopathy of prematurity causes visual impairment due to destructive neoangiogenesis after degeneration of the retinal microvasculature. This study was aimed at analyzi... More
Retinopathy of prematurity causes visual impairment due to destructive neoangiogenesis after degeneration of the retinal microvasculature. This study was aimed at analyzing whether local delivery of Semaphorin-3C (Sema3C) suppresses pathological retinal angiogenesis. Sema3C exerted potent inhibiting effects in cellular models of angiogenesis. In an endothelial cell xenotransplantation assay, Sema3C acted primarily on immature microvessels by inducing endothelial cell apoptosis. Intravitreal administration of recombinant Sema3C disrupted endothelial tip cell formation and cell-cell contacts, which led to decreased vascular bed expansion and vessel branching in the growing retinal vasculature of newborn mice, while not affecting mature vessels in the adult retina. Sema3C administration strongly inhibited the formation of pathological pre-retinal vascular tufts during oxygen-induced retinopathy. Mechanistically, Sema3C signaled through the receptors Neuropilin-1 and PlexinD1, which were strongly expressed on vascular tufts, induced VE-cadherin internalization, and abrogated vascular endothelial growth factor (VEGF)-induced activation of the kinases AKT, FAK, and p38MAPK. This disrupted endothelial cell junctions, focal adhesions, and cytoskeleton assembly resulted in decreased cell migration and survival. Thus, this study identified Sema3C as a potent and selective inhibitor of pathological retinal angiogenesis. Keywords: Sema3C; angiogenesis; retinopathy of prematurity; semaphorin. Less
ABSTRACT: The effects of mechanical cues on cell behaviors in 3D remain difficult to characterize as the ability to tune hydrogel mechanics often requires changes in the ... More
ABSTRACT: The effects of mechanical cues on cell behaviors in 3D remain difficult to characterize as the ability to tune hydrogel mechanics often requires changes in the polymer density, potentially altering the material’s biochemical and physical characteristics. Additionally, with most PEG diacrylate (PEGDA) hydrogels, forming materials with compressive moduli less than ∼10 kPa has been virtually impossible. Here, we present a new method of controlling the mechanical properties of PEGDA hydrogels independent of polymer chain density through the incorporation of additional vinyl group moieties that interfere with the cross-linking of the network. This modification can tune hydrogel mechanics in a concentration dependent manner from <1 to 17 kPa, a more physiologically relevant range than previously possible with PEG-based hydrogels, without altering the hydrogel’s degradation and permeability. Across this range of mechanical properties, endothelial cells (ECs) encapsulated within MMP-2/MMP-9 degradable hydrogels with RGDS adhesive peptides revealed increased cell spreading as hydrogel stiffness decreased in contrast to behavior typically observed for cells on 2D surfaces. EC-pericyte cocultures exhibited vessel-like networks within 3 days in highly compliant hydrogels as compared to a week in stiffer hydrogels. These vessel networks persisted for at least 4 weeks and deposited laminin and collagen IV perivascularly. These results indicate that EC morphogenesis can be regulated using mechanical cues in 3D. Furthermore, controlling hydrogel compliance independent of density allows for the attainment of highly compliant mechanical regimes in materials that can act as customizable cell microenvironments. KEYWORDS: vasculogenesis, tissue engineering, biomimetic material, PEG hydrogel Less
Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have g... More
Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM-MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion-induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM-MSCs. Seventy mice were pre-treated with BM-MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro-vascular endothelial cells (HPMVECs) were pre-conditioned with BM-MSCs by oxygen-glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI-treated mice, administration of BM-MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD-treated HPMVECs, co-culture with BM-MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM-MSCs decreased the level of PI3K class I and p-Akt while the expression of PI3K class III was increased. Finally, BM-MSCs-induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM-MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM-MSCs and will help to develop new cell-based therapeutic strategies in lung injury. Less
In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill ... More
In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention. Less
Japanese encephalitis virus (JEV) strains can be separated into 5 genotypes (g1 to g5) based on sequence similarity. JEV g5 strainshave been rarely isolated and are poorl... More
Japanese encephalitis virus (JEV) strains can be separated into 5 genotypes (g1 to g5) based on sequence similarity. JEV g5 strainshave been rarely isolated and are poorly characterized. We report here the full characterization of a g5 virus generated using a cDNA-based technology and its comparison with a widely studied g3 strain. We did not observe any major differences between those viruses when their infectious cycles were studied in various cell lines in vitro. Interestingly, the JEV g5 strain was highly pathogenic when inoculated to BALB/c mice, which are known to be largely resistant to JEV g3 infection. The study of chimeric viruses between JEV g3 and g5 showed that there was a poor viral clearance of viruses that express JEV g5 structural proteins in BALB/c mice blood, which correlated with viral invasion of the central nervous system and encephalitis. In addition, using an in vitro model of the blood-brain barrier, we were able to show that JEV g5 does not have an enhanced capacity for entering the central nervous system, compared to JEV g3. Overall, in addition to providing a first characterization of the understudied JEVg5, our work highlights the importance of sustaining an early viremia in the development of JEV encephalitis Less
Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progres... More
Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progression of stroke. Although it has been well documented that brain microvascular endothelial cells and astrocytes play a critical role in the maintenance of BBB integrity, pericytes, sandwiched between endothelial cells and astrocytes, remain poorly studied in the pathogenesis of stroke. Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus. Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody. The in vivo relevance of these findings was further corroborated in the stroke model of mice wherein the mice, demonstrated disruption of the BBB integrity and concomitant increase in the expression of VEGF in the brain tissue as well as in the isolated microvessel. These findings thus suggest the role of pericyte-derived VEGF in modulating increased permeability of BBB during stroke. Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease. Less
The NG2 proteoglycan stimulates the proliferation and migration of various immature cell types, including pericytes. However, the role of NG2 in mediating pericyte/endoth... More
The NG2 proteoglycan stimulates the proliferation and migration of various immature cell types, including pericytes. However, the role of NG2 in mediating pericyte/endothelial cell interaction has been less clear. In this study, we show that pericyte-specific NG2 ablation causes several structural deficits in blood vessels in intracranial B16F10 melanomas, including decreased pericyte ensheathment of endothelial cells, diminished formation of endothelial junctions, and reduced assembly of the vascular basal lamina. These deficits result in decreased tumor vessel patency, increased vessel leakiness, and increased intratumoral hypoxia. NG2-dependent mechanisms of pericyte interaction with endothelial cells are further explored in pericyte/endothelial cell co-cultures. siRNA-mediated NG2 knockdown in pericytes leads to reduced formation of pericyte/endothelial networks, reduced formation of ZO-1 positive endothelial cell junctions, and increased permeability of endothelial cell monolayers. We also show that NG2 knockdown results in loss of β1 integrin activation in endothelial cells, revealing a mechanism for NG2-dependent cross talk between pericytes and endothelial cells. Less
Cerebral amyloid angiopathy (CAA) is common in patients with Alzheimer's disease (AD) and may contribute to cerebral hemorrhage. We previously demonstrated that tissue pl... More
Cerebral amyloid angiopathy (CAA) is common in patients with Alzheimer's disease (AD) and may contribute to cerebral hemorrhage. We previously demonstrated that tissue plasminogen activator (tPA) and plasminogen (PLG) accumulated at the periphery of compact amyloid-cored plaques and in the walls of CAA-containing blood vessels in the brains of Tg2576 mice, a widely used AD mouse model. We had also observed that zinc-triggered tPA and PLG induction were observed in mouse cortical cultures. Because zinc also accumulates in amyloid plaques and blood vessel walls in AD brains, we examined whether zinc increases mRNA and protein levels of tPA and PLG in brain endothelial cells and pericytes. Four hours after the exposure of brain endothelial cells (bEnd.3) to 40 µM zinc, the mRNA and protein expressions of tPA and its substrate PLG were significantly increased. In the case of brain pericyte cultures, increases in tPA and PLG expression were also detected 2 hr after treatment. However, amyloid-β (Aβ)1-42 oligomers did not augment tPA and PLG expression in bEnd.3 cells and pericytes, suggesting that zinc but not Aβ induces tPA and PLG accumulation in CAA found in the AD brain. Less
Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the d... More
Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the determining factors for successful isolation of recombinant antibodies from libraries lies in the quality of the libraries i.e. the number of correctly folded, functional antibodies contained in the library. Here, we describe the construction of a novel, high quality, synthetic single domain antibody library dubbed Predator. The library is based on the HEL4 domain antibody with the addition of recently reported mutations concerning the amino acid composition at positions critical for the folding characteristics and aggregation propensities of domain antibodies. As a unique feature, the CDR3 of the library was designed to mimic the natural human immune response by designating amino acids known to be prevalent in functional antibodies to the diversity in CDR3. CDR randomizations were performed using trinucleotide synthesis to avoid the presence of stop codons. Furthermore a novel cycle free elongation method was used for the conversion of the synthesized single stranded DNA containing the randomized CDRs into double stranded DNA of the library. In addition a modular approach has been adopted for the scaffold in which each CDR region is flanked by unique restrictions sites, allowing easy affinity maturation of selected clones by CDR shuffling. To validate the quality of the library, one round phage display selections were performed on purified antigens and highly complex antigen mixtures such as cultured eukaryotic cells resulting in several specific binders. The further characterization of some of the selected clones, however, indicates a reduction in thermodynamic stability caused by the inclusion the additional mutations to the HEL4 scaffold. Less
Background: There is increasing evidence to suggest that pericytes play a crucial role in regulating the remodeling state of blood vessels. As cerebral pericytes are embe... More
Background: There is increasing evidence to suggest that pericytes play a crucial role in regulating the remodeling state of blood vessels. As cerebral pericytes are embedded within the extracellular matrix (ECM) of the vascular basal lamina, it is important to understand how individual ECM components influence pericyte remodeling behavior, and how cytokines regulate these events. Methods: The influence of different vascular ECM substrates on cerebral pericyte behavior was examined in assays of cell adhesion, migration, and proliferation. Pericyte expression of integrin receptors was examined by flow cytometry. The influence of cytokines on pericyte functions and integrin expression was also examined, and the role of specific integrins in mediating these effects was defined by function-blocking antibodies. Expression of pericyte integrins within remodeling cerebral blood vessels was analyzed using dual immunofluorescence (IF) of brain sections derived from the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Results: Fibronectin and collagen I promoted pericyte proliferation and migration, but heparan sulfate proteoglycan (HSPG) had an inhibitory influence on pericyte behavior. Flow cytometry showed that cerebral pericytes express high levels of α5 integrin, and lower levels of α1, α2, and α6 integrins. The pro-inflammatory cytokine tumor necrosis factor (TNF)-α strongly promoted pericyte proliferation and migration, and concomitantly induced a switch in pericyte integrins, from α1 to α2 integrin, the opposite to the switch seen when pericytes differentiated. Inhibition studies showed that α2 integrin mediates pericyte adhesion to collagens, and significantly, function blockade of α2 integrin abrogated the pro-modeling influence of TNF-α. Dual-IF on brain tissue with the pericyte marker NG2 showed that while α1 integrin was expressed by pericytes in both stable and remodeling vessels, pericyte expression of α2 integrin was strongly induced in remodeling vessels in EAE brain. Conclusions: Our results suggest a model in which ECM constituents exert an important influence on pericyte remodeling status. In this model, HSPG restricts pericyte remodeling in stable vessels, but during inflammation, TNF-α triggers a switch in pericyte integrins from α1 to α2, thereby stimulating pericyte proliferation and migration on collagen. These results thus define a fundamental molecular mechanism in which TNF-α stimulates pericyte remodeling in an α2 integrin-dependent manner. Less
Pericytes play critical roles in the development, maturation and remodeling of blood vessels, and in the central nervous system (CNS), evidence suggests that pericytes al... More
Pericytes play critical roles in the development, maturation and remodeling of blood vessels, and in the central nervous system (CNS), evidence suggests that pericytes also regulate blood flow and form an integral part of the blood-brain barrier. The study of this important cell type has been hampered by the lack of any pericyte-specific marker and by the difficulty of culturing pericytes in adequate numbers to high purity. Here we present a novel yet simple approach to isolate and culture large numbers of pericytes from the mouse CNS that nevertheless leads to very pure pericyte cultures. In our method, vascular cells obtained from adult mice brains are cultured initially under conditions optimized for endothelial cells, but after two passages switched to a medium optimized for pericyte growth. After growing the cells for 1-2 additional passages we obtained a largely homogeneous population of cells that expressed the pericyte markers NG2, PDGFβ-receptor, and CD146, but were negative for markers of endothelial cells (CD31), microglia (Mac-1) and astrocytes (GFAP). Under these conditions, pericytes could be grown to high passage number, and were maintained highly pure and largely undifferentiated, as determined by antigen expression profile and low levels of α-SMA expression, a marker of pericyte differentiation. Furthermore, switching the cells from pericyte medium into DMEM containing 10% FBS promoted α-SMA expression, demonstrating that high passage pericytes could still differentiate. Thus, we provide an alternative approach to the culture of CNS pericytes that is easy to establish and provides large numbers of highly pure pericytes for extended periods of time. This system should provide others working in the pericyte field with a useful additional tool to study the behavior of this fascinating cell type. Copyright © 2012 Elsevier Inc. All rights reserved. Less
Tumor vasculature is irregular, abnormal, and essential for tumor growth. Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels und... More
Tumor vasculature is irregular, abnormal, and essential for tumor growth. Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels under angiogenic conditions. As primary cells in culture, pericytes and EPC share many properties such as tube/network formation and response to kinase inhibitors selective for angiogenic pathways. Expression of cell surface proteins including platelet-derived growth factor receptor, vascular cell adhesion molecule, intercellular adhesion molecule, CD105, desmin, and neural growth proteoglycan 2 was similar between pericytes and EPC, whereas expression of P1H12 and lymphocyte function–associated antigen-1 clearly differentiates the cell types. Further distinction was observed in the molecular profiles for expression of angiogenic genes. Pericytes or EPC enhanced the invasion of MDA-MB-231 breast cancer cells in a coculture assay system. The s.c. coinjection of live pericytes or EPC along with MDA-MB-231 cells resulted in an increased rate of tumor growth compared with coinjection of irradiated pericytes or EPC. Microvessel density analysis indicated there was no difference in MDA-MB-231 tumors with or without EPC or pericytes. However, immunohistochemical staining of vasculature suggested that EPC and pericytes may stabilize or normalize vasculature rather than initiate vasculogenesis. In addition, tumors arising from the coinjection of EPC and cancer cells were more likely to develop lymphatic vessels. These results support the notion that pericytes and EPC contribute to malignancy and that these cell types can be useful as cell-based models for tumor vascular development and selection of agents that may provide therapeutic benefit. Less
